Generalized glucocorticoid resistance is associated with glucocorticoid receptor (GR, NR3C1) mutations. Three novel heterozygous missense NR3C1 mutations (R477S, Y478C and L672P) were identified in patients presenting with adrenal incidentalomas, glucocorticoid excess without Cushing syndrome. Dexamethasone (DXM) binding studies demonstrated that the affinity of GR_R477S and GR_Y478C mutants, located in the DNA-binding domain of GR, was similar to wild-type GR (Kd = 2-3 nM). In contrast, GR_L672P mutant, located in the ligand-binding domain of GR, was unable to bind glucocorticoids and was more sensitive to protein degradation. GR subcellular distribution revealed a marked decrease in DXM-induced nuclear translocation of GR_R477S and GR_Y478C mutants, whereas GR_L672P remained exclusively cytoplasmic. Chromatin immunoprecipitation demonstrated impaired recruitment of DBD mutants onto the regulatory sequence of FKBP5. Transactivation assays disclosed the lack of transcriptional activity of GR_R477S and GR_L672P while GR_Y478C had a reduced transactivation capacity. Three-D modeling indicated that R477S lost two essential hydrogen bonds with DNA, Y478C resulted in altered interaction with surrounding amino-acids, destabilizing DBD, while L672P altered the H8 helix folding, leading to unstructured LBD. This study identifies novel NR3C1 mutations with their molecular consequences on altered GR signaling and suggests that genetic screening of NR3C1 should be conducted in patients with subclinical hypercorticism.